This goal of this project is the elucidation of mechanisms relating dietary lipids as well as other nutrients and dietary factors to mechanisms of membrane signaling. Experiments using human breast cancer cells (MCF-7) to examine the role of membrane lipids in the generation of signaling lipid mediators have generated significant information in two areas. First, MCF- 7 cells lack the ability to synthesize ether-linked phospholipids. The deficiency results from the failure of the alkyl dihydroxy acetone phosphate synthetase enzyme system to use fatty alcohol as a precursor for the ether-linked lipids. This cellular system represents a model to study the cellular physiology of ether-linked lipids. Second, multidrug resistant MCF-7 cells show an increase in phospholipase D activity (about 4x over drug sensitive controls) in response to phorbol ester. Pyrroline 5-carboxylate, a physiologic mediator of redox transfers, appears also to be a potent stimulator of phospholipase D. Gas chromatography/mass spectrometry is being used to characterize the phospholipase D response in the tumorigenic MCF-7 cells and in a "normal," nontumorigenic human breast cell line. These studies are expected to define the role that lipid mediators, such as phosphatidic acid and diacylglycerol, play in regulating mitogenic responses of breast cells. The impact of dietary fat, simulated by experimentally modifying the cellular membrane lipid composition, will be investigated with regard to the regulation of phospholipase-generated lipid mediators. Collectively, results from these experiments will elucidate the function of membrane lipids in cellular growth control. Future studies will emphasize the effects of trace fatty acids and fatty acid conjugates.